The mouse t-complex is a region of chromosome 17 in which there maps a number "lethal t-mutations" that, when homozygous, cause disturbances of early development leading ultimately to the death of the embryo. The main obstacle to the study of lethal t-mutant gene expression during embryogenesis has been the difficulty of obtaining appropriate experimental material. Recently we have devised a method of cell culture that enables us to establish pluripotent embryonic stem (ES) cell lines directly from early mouse embryos. Since each of these cell lines constitutes a virtually unlimited supply of cells from a single early embryo of a particular genotype, they can be used to circumvent many of the difficulties inherent in studies of intact early mouse embryos. We have already been able to use this method to establish ES cell lines that are either homozygous (t/t) or heterozygous (+/t) for one of the lethal t-mutations, tw5. We now propose to establish t/t and +/t ES cell lines from embryos carrying two different lethal t-mutations, t0 and t9. All of these cell lines will then be used in studies aimed at identifying the products of lethal t-mutant gene expression in ES cells. One approach will be to use a sophisticated computer-analyzed system of 2D gel electrophoresis to compare the patterns of protein synthesis in t/t and +/t with those of +/+ cells. This should enable us to identify polypeptides that might result from lethal t-mutant gene expression. Further studies will be aimed at determining which of these are lethal t-mutation-specific. In addition, we will test the hypothesis that lethal t-mutations cause alterations in the expression of cell surface molecules of early embryonic cells. t/t ES cells will be used as antigens to raise monoclonal antibodies that detect molecules present on the surface of t/t ES cells as a consequence of lethal t-mutant gene expression. Our immediate goal is to learn more about the nature of the lethal t-mutant gene products, as a first step toward understanding their function. Ultimately such information should help to elucidate the mechanism of the control of normal embryonic development. The results could have implications for research on human development since there are three examples of possible t-complex analogues in man.